Luminescent iridium(III)–peptide bioconjugates for bioanalytical and biomedical applications

Abstract

Iridium(III) complexes are alternative bioimaging probes due to their tunable photophysical properties, but are limited by poor cell penetrability and high cytotoxicity. Recently, iridium(III)–peptide bioconjugates have received significant attention as bifunctional molecules in bioanalytical and biomedical fields. Conjugation to peptides endows iridium(III) complexes with specificity, potentially overcoming the side effects and drug resistance of metallodrugs, whilst enhancing cellular uptake due to the improved cell penetrability, low cytotoxicity and targetability of peptides. In this review, we briefly introduce the interactions between iridium(III) complexes and amino acids/peptides, including coordination to amino acids and detection and/or inhibition of peptides. We describe imaging applications of iridium(III)–peptide bioconjugates, involving direct coordination of functional peptides or ligand modification, for targeted imaging. Next, we present therapeutic and theranostic applications of iridium(III)–peptide bioconjugates through targeting of DNA and proteins. Finally, we outline the challenges and future opportunities in the development of iridium(III)–peptide bioconjugates for precision medicine.

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Xiaolei Wu (left 1), Jing Wang (left 2), Wanhe Wang (middle), Jianxiong Du (right 2) and Shaozhen Jing (right 1)

Shaozhen Jing is a master's student in the Institute of Medical Research at the Northwestern Polytechnical University, and his research interests focus on the development of luminescent sensing platforms for disease-related analytes.

Xiaolei Wu is a master's student in the Institute of Medical Research at the Northwestern Polytechnical University, and her research interests focus on the development of luminescent sensing platforms for disease-related analytes.

Daniel Shiu-Hin Chan

Daniel Shiu-Hin Chan completed his PhD degree in Chemistry at the University of Cambridge, UK, and his BSc degree at The University of New South Wales, Australia. He is currently appointed as a Research Associate at the City University of Hong Kong under Prof. Alex Chun Yuen Wong.

Sang-Cuo Nao

Sang-Cuo Nao is a PhD student at the University of Macau, focusing on cancer theranostics through metal-complex development. She also researches luminescent high-throughput sensing for drug screening and hazardous compound detection for quality control and food safety. Her work aims to advance medical science, improve drug discovery, and safeguard public health.

Jianxiong Du is a master's student in the Jiangxi Provincial Key Laboratory of Functional Molecular Materials Chemistry at the Jiangxi University of Science and Technology, and his research interests focus on the development of luminescent sensing platforms for disease-related analytes.

Chun-Yuen Wong

Chun-Yuen Wong received his BSc in 2000 and PhD in 2005 from the University of Hong Kong. He was a Croucher Foundation Postdoctoral Fellow at Harvard University from 2005 to 2006. He joined the City University of Hong Kong in November 2006. His current research interests include: (1) probing the metal–carbon bonding interactions in organometallic complexes through spectroscopic and theoretical means in order to provide insight on the design of functional organometallic complexes; (2) designing metal/semiconductor hybrid nanostructures for photonic application.

Jing Wang obtained her PhD degree at Sun Yat-sen University, then undertook her postdoctoral research in the group of Prof. Dennis Lo at the Chinese University of Hong Kong. She is currently working as an Associate Professor at the Institute of Medical Research at the Northwestern Polytechnical University. Her research interests include the development of visual sensing platforms based on nucleic acid amplification and gold nanoparticles for non-invasive disease diagnosis.

Chung-Hang Leung

Chung-Hang Leung completed his PhD in 2002 at the City University of Hong Kong. After completing a five-year post-doctoral fellowship at the Department of Pharmacology, Yale University, he was appointed as a Research Assistant Professor at the University of Hong Kong and then at the Hong Kong Baptist University. He is currently working as a Professor at the University of Macau. His primary research interests are in structure-based drug discovery and in the development of oligonucleotide-based drug-screening methods.

Wanhe Wang obtained his MSc degree at Jilin University, then completed his PhD degree at the Hong Kong Baptist University. He is currently working as Associate Professor at the Institute of Medical Research at the Northwestern Polytechnical University. His research interests include the development of luminescent sensing platforms for disease-related biomolecules and metallodrugs.

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Introduction

Over the past few decades, transition metal complexes have been extensively employed in organic light-emitting diodes, light-emitting electrochemical cells, and as luminescent labels or sensors, due to their interesting photochemical and photophysical properties.^1–3 Transition metal complexes have tunable emission wavelengths, high luminescence quantum yields, and relatively long emission lifetimes.⁴ In particular, iridium(III) complexes have been used for bioimaging, photodynamic therapy (PDT), and targetable therapy, due to their tunable structures, photocatalytic activity, large Stokes shifts, facile cellular uptake, and organelle-targeting properties.^4–13 However, iridium(III) complexes are often limited by their low solubility, cytotoxicity, and low cell penetration, hindering their biological applications.^14,15

Peptides are composed of multiple amino acids joined by amide bonds, and show versatile functions in various aspects of biological functions, such as cancer cell targetability and subcellular localization.^16,17 Considerable efforts have been made towards the development of peptide molecules with precisely tailored biological and structural properties over the past decades.^18–21 Meanwhile, peptides have also received intensive attention for analytical and therapeutic applications in the past two decades.^22–25 Peptides occupy an intermediate chemical space between small organic molecules and large biologics for drug design and discovery.^26,27 In drug discovery, peptide-based molecules offer advantages of high biological activity, high specificity, and low toxicity.²⁸ However, peptides can suffer from low stability and lack of oral bioavailability, limiting their pharmaceutical potential.²⁹ Hence, peptide conjugation has become a popular strategy for the development of clinically relevant therapeutic agents.^30,31

Compared with other transition metal complexes, iridium(III) complexes have distinct luminescence properties that make them attractive as bioimaging probes.^5,7,32,33 For example, iridium(III) complexes show intense phosphorescence at room temperature with a long emission lifetime, while complexes of rhodium(III), which is also in group 14, only give measurable emission at low temperatures, consistent with stronger spin–orbit coupling of iridium(III) relative to rhodium(III).³⁴ Moreover, the emission color of iridium(III) polypyridine complexes can be readily tuned from bluish-green to near-infrared (NIR) through the use of different cyclometalated/ancillary ligands, while emissive ruthenium(II) polypyridine complexes are mostly confined to the orange to red region.^35,36 Furthermore, most iridium(III) complexes exhibit high intrinsic mitochondrial specificity due to their cationic and lipophilic properties, and can be modified to target other organelles with high specificity, while ruthenium(II) complexes generally show lower lipophilicity and poor membrane permeability.^37,38

However, iridium(III) complexes can sometimes display poor cell penetrability and high cytotoxicity, preventing their further application in biological systems.³⁹ On the other hand, peptides have low cytotoxicity, good targetability and/or high cell penetrability. Many functional peptides have been applied in peptide–drug conjugates (PDCs) to potentially improve drug targeting, reduce side effects in other cells, and improve drug bioavailability.^29,40 Additionally, certain peptide sequences, both natural and synthetic, have specific receptors that are overexpressed in cancer cells compared with normal cells, which can be exploited for targeted drug delivery.^41,42 Therefore, iridium(III)–peptide bioconjugates could successfully combine the advantages of iridium(III) complexes and peptides, while overcoming the cytotoxicity of the iridium(III) complex.

Furthermore, the introduction of cationic and lipophilic iridium(III) complex can overcome another drawback of peptides: their low metabolic stability and poor oral bioavailability.^43,44 In the field of peptide chemistry, certain strategies for improving the stability of peptides have been explored, such as cyclization, increasing the steric bulk of side chains, D-amino acids, N-acetylation and C-amidation (N-methylation, modification in the end of peptide by polyethylene glycol) or adding fatty chains.^45,46 Thus, conjugation with iridium(III) complexes could provide an alternative method to improve peptide stability, complementing existing techniques.^45,47–49

A few strategies are available for synthesizing iridium(III)–peptide conjugates. Generally, peptides can be first synthesized either through solid-phase peptide synthesis or solution-phase peptide synthesis. Subsequently, peptides can be conjugated to iridium(III) complexes by amide bond formation, click chemistry, or coordination to the side chain of amino acid residues (e.g. imidazole in histidine).⁵⁰ Amide bond formation is a classical reaction that is widely used in all areas of organic chemistry.⁵¹ Click chemistry is a relatively newer strategy that can be used for preparing more structurally challenging bioconjugates and for efficiently establishing a library of bioconjugates.^52,53 For some peptides that have specific amino acids such as histidine, the coordination strategy could be preferable for rigidifying the peptide for better affinity.

A variety of iridium(III) complexes functionalized with bioactive peptides have been extensively applied in cell imaging, drug discovery and other areas.^54–56 However, although a few reviews describing metal–peptide bioconjugates have been published,^{44,51,57–61} specific reviews on the bioanalytical and biomedical applications of iridium(III)–peptide bioconjugates are scarce. In this review, we summarize the interactions between iridium(III) complexes and peptides and the emerging applications of iridium(III)–peptide bioconjugates in biomedical fields (Scheme 1), highlighting their widespread application for targeted luminescent imaging and as therapeutic agents. However, we exclude examples of using peptides as a linker to construct iridium(III) complexes, as the peptide moiety only plays a minor role in such complexes.⁶² We also outline the challenges and future opportunities in the development of iridium(III)–peptide bioconjugates and applications for precision medicine.

Scheme 1 Overview of luminescent iridium(III)–peptide bioconjugates for bioanalytical and biomedical applications.

Interactions between iridium(III) complexes and peptides

The development of iridium(III)–peptide bioconjugates was stimulated by early work exploring the interactions of iridium(III) complexes with peptides, whilst taking cues from the established field of peptide-based targeted drug delivery.⁴⁵ Peptide-based targeted drug delivery, including antibody–drug conjugates (ADC), has been considerably studied due to its receptor specificity, providing a basis for exploring iridium(III)–peptide bioconjugates.⁶³

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder, with over 51.6 million individuals with AD-related dementias worldwide in 2019 and nearly 9.83 million individuals with AD in China in 2020.⁶⁴ One main hallmark of AD is the aggregation of amyloid-β (Aβ) peptides in the brain of AD patients.^65,66 Aβ is a native metal-binding peptide with a typical N-terminal metal-binding sequence including three histidines,⁶⁷ and copper dyshomeostasis was also observed in AD patients.⁶⁸ These studies inspired the application of transition metal complexes for interacting with amino acids and Aβ. In 2008, a landmark study by Ma's group applied a solvento iridium(III) complex 1 for the detection of histidine and histidine-rich proteins.⁶⁹ The labile solvento ligands of complex 1 could be replaced by histidine to form iridium(III)–peptide conjugates, similar to the interaction between Aβ and copper ions. After this, the same group further synthesized two solvento iridium(III) complexes 2 and 3 with water (H₂O) ligands as luminescent probes for Aβ_1–40 peptide and as inhibitors of amyloid fibrillogenesis (Fig. 1), which detected and inhibited Aβ_1–40 through the replacement of labile H₂O ligands by the imidazole N-donor moiety of histidine residues of the Aβ_1–40 peptide to form a conjugate.⁷⁰ Moreover, other transition metal complexes, such as copper(II), platinum(II), and ruthenium(II) complexes, have also been found to inhibit the aggregation of Aβ peptides.⁷¹

Fig. 1 Chemical structures of complexes 1–8.

Ma's group further reported that iridium(III) complexes with diverse ligands could inhibit Aβ_1–40 peptides via non-covalent interactions.⁷² They synthesized a series of iridium(III) complexes based on previous C^N ligands, and demonstrated that the interactions between iridium(III) complexes and peptides were not confined to solvento iridium(III) complexes. The top candidate complex 4 was identified as a dual imaging and modulating probe of Aβ for the treatment of AD.

Cyclometalated iridium(III) complexes can also be used as photosensitizers due to their ability to generate reactive oxygen species (ROS) under light irradiation.^73–75 ROS could be produced through an electron transfer (˙OH, O₂˙⁻)-based type I pathway or an energy transfer mechanism (¹O₂)-based type II pathway.^76–78 In 2016, Lim's group reported that complex 5 with a dimethylamino group on the N^N ligand could modulate amyloidogenic peptide aggregation through photooxidation via¹O₂ generation.⁷⁹ The same group also found that the fluorine-substituted 2-phenylquinoline-based solvento complex 6 showed good ability to interact with Aβ peptide in a coordination-dependent manner like previously reported solvento iridium(III) complexes as well as through photooxidation (Fig. 1).⁸⁰ Very recently, Lee's group reported that a boron-dipyrromethene-based iridium(III) complex 7 can oxidize amyloidogenic peptides through both type I and type II processes.⁸¹ The introduction of the boron-dipyrromethene moiety overcomes the drawbacks of weak absorbance in the short wavelength region and transient triplet excited states, indicating that the combination of organic dyes and iridium(III) complexes can improve the PDT performance of the complexes.

Apart from histidine, solvento iridium(III) complexes have also been found to coordinate with glutamine (Gln). In 2016, Mao's group designed solvento iridium(III) complex 8 featuring an aldehyde group for the selective detection and imaging of Gln in live cells,⁸² but interactions between iridium(III) complexes and Gln are less studied (Table 1). Other examples of using iridium(III) complexes as photocatalysts for modifying bioactive peptides are out of the scope of this review.^83,84 In the following sections, we focus on the conjugation of iridium(III) complexes with functional peptides to generate bifunctional iridium(III)–peptide bioconjugates, similar to the well-known ADC and proteolysis-targeting chimera (PROTAC) that have received significant attention for bioanalytical and biomedical applications in recent years.⁸²

Table 1 Photophysical properties of complexes 1–8

Complex	Target	Solvent	λ abs/nm	λ emi/nm	Φ PL	Φ Δ	Lifetime/ns	Ref.
1 (H2O/CH3CN)	Histidine/histidine-rich proteins	—	—	—	—	—	—	69
2	Aβ1–40 fibrils/Aβ1–40 monomers	—	—	—	—	—	—	70
3	Aβ1–40 fibrils/Aβ1–40 monomers	—	—	—	—	—	—	70
4	Aβ1–40 fibrils/Aβ1–40 monomers	PBS	280, 422	586	0.0806	—	4.502	72
5	Aβ peptides	HEPES	463 (H2O)	600	0.41	0.25	238	79
6	Aβ peptides	H2O	274, 336, 446	589	0.0071	—	4.8	80
7	Aβ peptides	CH2Cl2	505	563 (H2O)	0.1738	—	—	81
8	Gln	DMSO/PBS	281, 318, 420	557	0.04	—	100	82

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Targetable imaging applications of iridium(III)–peptide bioconjugates

Early studies on the interaction between iridium(III) complexes and peptides prompted the development of iridium(III) complex–peptide bioconjugates. In 2011, inspired by Ma's work, Fei's group investigated the coordination mechanism of [Ir(ppy)₂(CH₃CN)₂]OTf (ppy = 2-phenylpyridine, OTf = triflate) with histidine and histidine-derived peptides.⁸⁵ The peptide sequence modified by an N-terminal single histidine or a dihistidine (dH) short motif could be specifically labeled by the solvento iridium(III) complex (Fig. 2). These highly efficient labeling reactions can be performed at room temperature and are largely unperturbed by other amine groups from nearby lysine residues or from the N-terminus. The investigators further applied this labeling strategy for cell imaging, and designed a cell-penetrating peptide (CPP) with a histidine residue at the N-terminus, thus generating an iridium(III) complex-labeled CPP. Furthermore, they designed a dual-functional peptide with both CPP and a mitochondria-targeting sequence (MTS) (Met-Leu-Ala-Lys-Gly-Leu-Pro-Pro-Lys-Ser-Val-Leu-Val-Lys-Gly-Gly-His-His-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg) with a dH motif, thus resulting in the construction of the bioconjugate 9 as a dual-functional peptide. This bioconjugate can effectively image cytoplasm and mitochondria, respectively, due to the introduction of the targeting peptides. This work provides additional options for preparing peptide conjugation through coordination to the metal center, instead of the usual covalent cross-linking through the amine group of the N-terminus or a lysine residue, or the thiol group of a cysteine residue.

Fig. 2 (A) Two peptide labeling methods. (B) Structures of bioconjugates 9–12.

Later on, Fei's group investigated the distance between histidines for cyclic formation.⁸⁶ They found that two or three amino acids gave robust results, generating bioconjugates 10 and 11, respectively (Fig. 2). Based on this result, they developed the iridium(III) bioconjugate 12, containing the coordination-based cyclized functional peptide, cRGD, for cancer cell targeting. This method allows a convenient and convincing assessment of the cell selectivity of therapeutic peptide compounds. This work elegantly utilized iridium(III) coordination to rigidify peptide conformation, and amplify the biological activities of peptides.

Instead of coordination, the amide coupling strategy has been widely applied to generate iridium(III)–peptide conjugates. In 2011, Leeuwen's group synthesized peptide-functionalized bioconjugates 13–15 for the lifetime imaging of chemokine receptor 4 (CXCR4) expression using traditional amide bond formation strategies (Fig. 3).⁸⁷ The CXCR4 is a G-protein-coupled membrane receptor, which plays an important role in tumor progression and metastasis, and is thus emerging as a diagnostic and targetable delivery target.^88,89 The peptide Ac-Tz14011 has been identified as antagonist for CXCR4. Thus, 2-phenylpyridine-based triscyclometalated iridium(III) bioconjugates 13–15 bearing different numbers of Ac-Tz14011 were synthesized by forming an amide bond between the iridium(III) complex and the peptides. For the design of peptide–iridium(III) complex conjugates, this group initially investigated the crystal structure of CXCR4 complexed with the Ac-TZ14011 analogue to confirm that the luminescent iridium(III) label did not interfere with receptor binding, as the conjugation site in the peptide (d-Lys8) is distant from the pharmacophore. All three iridium(III)–peptide conjugates could visualize CXCR4 expression in CXCR4-transfected MDA-MB-231 cells (Fig. 3), and the trimeric conjugate 15 showed the best K_D of 66.3 nM and ratio of MFIRs (mean fluorescence intensity ratio) of 1.72. The long lifetime of bioconjugate 14 was also used for fluorescence lifetime imaging (FLIM) of CXCR4. In the cell viability experiment, bioconjugate 13 displayed low toxicity at 10 μM, whereas 14 and 15 were considerably more toxic. The reduced viability of derivatives 14 (+10 charge) and 15 (+15 charge) could be the result of the high positive charge, compared with Ac-Tz14011 with +5 charge. This is the first example of a neutral iridium(III) complex functionalized with peptides for specific targeting of a cancer-associated membrane receptor, but its application in native CXCR4-expressing cancer cells was not explored.

Fig. 3 (A) Chemical structure of bioconjugates 13–15. (B) Confocal microscopy and transmission images of the peptide-conjugated iridium(III) complexes on MDAMB231^CXCR4+ cells: (A) 1 μM of 13; (B) 1 μM of 14; (C) 1 μM of 15. Reproduced with permission from ref. 87. Copyright 2011, John Wiley and Sons.

Cationic cyclometalated iridium(III) complexes can be easily synthesized using a modular strategy.^90–92 First, chloro-bridged iridium(III) dimer precursors are prepared via the reaction of cyclometalated C^N ligands and iridium(III) chloride, which are then reacted with the appropriate ancillary N^N ligand to prepare the iridium(III) complexes.⁹³ Typically, the cyclometalated C^N ligands are used to modulate the photophysical properties of the bioconjugates, while ancillary N^N ligands are used to tether peptides.³⁴ In 2018, Ma's group developed a permanent formyl peptide receptor 2 (FPR2) imaging probe 16, which conjugated a WKYMVm (FPR2 agonist) to the ancillary ligand of an iridium(III) complex (Fig. 4).⁹⁴ Formyl-peptide receptors (FPRs) are therapeutic targets for a variety of diseases, including cancer, inflammation and neurodegenerative disease.⁹⁵ Bioconjugate 16 was synthesized by a standard solution-phase peptide protection and deprotection strategy. Bioconjugate 16 showed a maximum emission at 576 nm with a maximum excitation at 291 nm in DMSO. The probe imaged FPR2-expressing HUVEC cells in a dose- and time-dependent manner, and selectively targeted FPR2 in HUVEC cells, and also inhibited lipoxin A4 (LXA4)-triggered cell migration in HUVEC cells, thereby serving as a theranostic probe.

Fig. 4 Chemical structures of bioconjugates 16–22.

In 2017, Marchán's group reported a bioconjugate 17 to a tumor-targeting vector based on octreotide (OCT) peptide, (D-Phe)-Cys-Phe-(D-Trp)-Lys-Thr-Cys-Thr (Fig. 4).⁹⁶ The attachment of the iridium(III) complex to OCT was achieved through the formation of an amide bond between the carboxylic group in the benzimidazole diimine ligand and the N-terminal end of the peptide sequence through a stepwise solid-phase strategy. Bioconjugate 17 accumulated in cancer cells overexpressing somatostatin subtype-2 receptor (SSTR2), and the participation of the receptor was confirmed by competitive experiments. In cell imaging, bioconjugate 17 allowed the visualization of luminescent vesicles in the cytoplasm, most likely in endosomes, confirming the cellular uptake of the bioconjugate 17 in HeLa cells. Further imaging reflected a slightly lower cellular accumulation in MDA-MB-231 cells compared with that of HeLa cells as indicated by a reduced intensity of the luminescence signal. Overall, these results demonstrate that the design of iridium(III)–peptide conjugates can improve tumor selectivity. Moreover, this study highlighted the importance of a spacer (ethylene glycol flexible chain) to keep the metal complex away from the pharmacophore sequence and the β-turn peptide structure, which are key elements for recognition and binding to the receptor.

Ma's group also developed a gastrin-releasing peptide receptor (GRPr) imaging probe using a GRPr peptide antagonist, the statine-based JMV594 [(D-Phe)-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH₂] (Fig. 4).⁹⁷ Bioconjugate 18 was synthesized through a standard solution-phase peptide protection and deprotection strategy. 6-Aminohexanoic acid was used as a long-chain linker to combine the iridium(III) complex with JMV594, in order to reduce the likelihood of the iridium(III) complex interfering with the recognition ability of JMV594. Bioconjugate 18 exhibits an intense absorption band at 250–310 nm in CH₃CN and shows an excitation maximum at 321 nm and an emission maximum at 596 nm in DMSO, with a large Stokes shift of 275 nm. Bioconjugate 18 displayed negligible cytotoxicity against A549 cancer cells and the normal human hepatic cell line LO2 cells. In contrast, the iridium(III) complex without the peptide exhibited high toxicity against both A549 cells with an IC₅₀ of 7.31 μM, and moderate toxicity against LO2 cells with of an IC₅₀ of 27.75 μM. Thus, the incorporation of JMC594 abolishes the toxicity of the iridium(III) complex owing to reducing undesirable non-specific binding to biomolecules. Finally, bioconjugate 18 displayed negligible luminescence in living LO2 cells, but stronger emission in GRPr-expressing A549 cancer cells, which shows the potential of the probe for the diagnosis of cancer.

Organelle-specific probes are key to studying biological processes at the subcellular level.^98,99 Iridium(III)–peptide bioconjugates have been developed for specific targeting of organelles via conjugation with suitable peptides. In 2012, Klimant's group developed bioconjugates 19–21 as cellular O₂ sensors (Fig. 4).⁵⁴ Attaching different short peptide sequences to the iridium(III)-octaethylporphyrin provided different targeting abilities via simple ligand-exchange reactions by histidine residues. Specifically, bioconjugate 19 possessed histidine-tetraarginine with +9 charge, bioconjugate 20 possessed a truncated fragment of cell-penetrating bactenecin 7 peptide, PRPLP, with +3 charge, while bioconjugate 21 carried the RGD sequence, a tumor-cell-targeting vector, with +1 charge. Absorption and emission spectra of the bioconjugates were found to be similar. The complexes showed a Soret band at around 386 nm, Q-bands at 506 and 540 nm, and peptide absorption in the UV region. Their emission was at around 654 nm and they also showed unquenched lifetimes above 40 μs in PBS. Both 19 and 20 demonstrated efficient staining of MEF cells, accumulating in the perinuclear regions and partially colocalizing with endoplasmic reticulum (ER) Tracker Green, a marker for the ER. Moreover, positive cytoplasmic staining was observed for all of these cell lines, including COS-7, HeLa, SH-SY5Y, and PC12 cells and mixed cultures of primary neurons and astrocytes. However, bioconjugate 21 carrying the RGD peptide unexpectedly did not display specific staining in HeLa and SH-SY5Y cells. This indicates that it is possible for metal conjugation to abolish the binding functionality of RGD peptides, possibly due to steric interference, the hydrophobicity of the porphyrin core, or the positive charge of the iridium(III) ion. As a result, when designing bioconjugates of this kind, it is crucial to consider the impact of the iridium(III) moiety on the activity of the peptide.

In 2019, Pope's group reported a cationic cyclometalated iridium(III)–peptide bioconjugate 22, which linked a nuclear localization signal (NLS) peptide (PAAKRVKLD) into the ancillary ligand (Fig. 4).¹⁰⁰ To generate the PAAKRVKLD sequence, Fmoc-solid-phase peptide synthesis was used. The NLS peptide originates from human C-MYC protein and was first identified in 1988 to be essential for the nuclear localization of the protein, and three of its residues are cationic at physiological pH. Bioconjugate 22 had similar photophysical properties to the parent complex, which showed maximum emission at 677 nm in dilute aerated aqueous solvent and a lifetime of 38 ns. Imaging studies showed that incubation with 80–100 μM bioconjugate 22 promoted good cell uptake and nuclear localization in human fibroblast cells. In comparison, a structurally related, photophysically analogous iridium(III) complex lacking the peptide sequence showed very different biological behavior, with no evidence of nuclear, lysosomal or autophagic vesicle localization and significantly increased toxicity to the cells at concentrations >10 μM with induced mitochondrial dysfunction. Thus, it can be concluded that the NLS peptide successfully endows the translocation and nuclear uptake properties of 22, whilst lowering toxicity. This study demonstrates the use of subcellular targetable units to modulate the intracellular localization behavior of iridium(III) complexes.

In 2020, Lo's group also reported a organelle-targeting peptide-conjugated iridium(III) complex 23 through a perfluorobiphenyl (PFBP) clickable moiety, which is easily bioconjugatable with a thiol group for bioimaging and phototherapeutic applications (Fig. 5).¹⁰¹ Complex 23 showed a maximum emission at 577 nm with a maximum excitation at 350 nm, and a lifetime of 0.40 μs in PBS and CH₃CN (1/1). Taking advantage of the high reactivity and unique chemo- and regioselectivity offered by the π-clamp sequence, the reaction between the PFBP complexes with cysteine-containing peptides containing the π-clamp sequence afforded luminescent conjugates with desirable photophysical and photochemical characteristics and differential cellular uptake and localization properties in HeLa cells. This strategy enables easy preparation of structurally diverse iridium(III)–peptide bioconjugate, and offers the possibility of the design of new theranostic agents.

Fig. 5 Modification of PFBP compound with peptides through the π-clamp-mediated cysteine conjugation. Reproduced with permission from ref. 101. Copyright 2020, American Chemical Society.

Bimetallic molecules offer the possibility of improving solubility and enhancing biological activities. In 2020, Gimeno's group reported a luminescent bimetallic iridium(III)/Au(I)–peptide bioconjugate 24 as a potential theranostic agent, which introduced an bioactive Au(I) complex onto the side chain of the peptide, Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu), which binds to the opiate receptors in the central nervous system (Fig. 6).¹⁰² Some carcinomas, such as colorectal and pulmonary cancer, contain high levels of opioid peptides and their corresponding membrane-bound opioid receptors.¹⁰³ Thus, linkage of drugs to opioid peptides could enhance cancer cell recognition and cellular uptake. The pentapeptide was synthesized by solid-phase peptide synthesis, then linked to the iridium(III) fragments via an amide bond using a carboxylic acid-functionalized diimine ligand. Finally, alkynyl groups were chosen as handles for insertion of the Au fragment. The bioconjugate 24 showed a maximum emission at 613 nm with a maximum excitation at 375 nm in DMSO. The probe 24 showed a red-shifted emission at around 615 nm, which was attributed to the electron-withdrawing amide substituent that stabilizes the π* orbitals of the diamine. However, this bioconjugate had no cytotoxicity against A549 cells, which is not common among Au(I) complexes, which was attributed to the inability of cationic complexes to escape the lysosome. In cell imaging, the bioconjugate 24 was not localized to the mitochondria in A549 cells, and localized in lysosomes with Pearson coefficients ranging from 0.8–0.9. This result was also unexpected, as some iridium(III)/Au(I) complexes generally have mitochondria targetability. Possibly, the presence of protonatable basic substituents could facilitate lysosomal localization instead. This work opens the door for the development of bimetallic peptide bioconjugates for biomedical applications, but their diagnostic potential was not explored.

Fig. 6 Chemical structures of bioconjugates 24–26.

Inspired by the aggregation of Aβ peptides, FF-rich peptides have self-assembling ability, and are widely applied for enhancing cell penetration, bioimaging and therapeutic performance.¹⁰⁴ Recently, Chao's group developed a self-assembling bioconjugate 25 through conjugating the naphthalene-Phe-Phe-Lys moiety to C^N ligands, to achieve long-term lysosome imaging (Fig. 6).¹⁰⁵ The triphenylamine group on the N^N ligand enabled bioconjugate 25 to display aggregation-induced emission (AIE) features, generating emission at around 600 nm in H₂O. Further experiments found that bioconjugate 25 exhibited pH-responsive self-assembly due to π–π interactions among the naphthalene and phenylalanine units. This stronger interaction leads to regular-shaped nanoparticles at pH 7, while a weaker π–π stacking at pH 4 triggers the molecular self-assembly, forming irregularly shaped networks. Moreover, 25 successfully captured the lysosome dynamic response to mitochondria in HeLa cells through structured illumination microscopy (SIM).

Very recently, our group reported the first prostate-specific membrane antigen (PSMA) turn-on bioconjugate 26 (Fig. 6).¹⁰⁶ Lys-urea-Glu, a PMSA inhibitor, was conjugated into iridium(III) complex.^107,108 The bioconjugate 26 exhibited maximum excitation at 330 nm and a broad emission band with a maximum peak at around 670 nm in CH₃CN. This peptide sequence exhibits excellent properties such as small size, high aqueous solubility, high affinity for PMSA (in the low nanomolar range), good biocompatibility and specificity. The luminescence emission lifetime of 26 was determined to be 24 ns (τ₁) and 375 ns (τ₂) in CH₃CN. The binding affinity of 26 to PMSA was determined to be 9.49 nM, which is over 1000-fold lower than that of the positive control DUPA (12.4 μM). Moreover, 26 showed no obvious toxicity against the human prostatic carcinoma cell line LNCaP cells (IC₅₀ > 50 μM) and the normal cell line LO2 (IC₅₀ > 50 μM). In contrast, the parent complex without modification with Lys-urea-Glu was highly toxic to LNCaP cells (IC₅₀ = 0.082 μM) and LO2 cells (IC₅₀ = 0.50 μM). Furthermore, 22Rv1 tumors could be clearly visualized by probe 26 in a mouse xenograft model of prostate cancer. This is the first example of the in vivo use of an iridium(III)–peptide bioconjugate (Table 2).

Table 2 Photophysical properties of bioconjugates 9–26

Bioconjugate	Peptide	Target	λ abs/nm	λ emi/nm	Φ PL	Lifetime/ns	Ref.
9	CPP/MTS	Cell-penetrating/cell-crossing	—	—	—	—	85
10	—	—	—	—	—	—	86
11	—	—	—	—	—	—	86
12	cRGD	Cancer cell	—	—	—	—	86
13	Ac-Tz14011	CXCR4	—	—	—	—	87
14	Ac-Tz14011	CXCR4	—	—	—	—	87
15	Ac-Tz14011	CXCR4	—	—	—	—	87
16	Gly-Met-Val-Met-Try-Lys-Trp	FPR2	255 (CH3CN)	576 (DMSO)	—	4620	94
17	(D-Phe)-Cys-Phe-(D-Trp)-Lys-Thr-Cys-Thr	SSTR2	—	—	—	—	96
18	(D-Phe)-Gln-Trp-Ala-Val-Gly-His-Sta-Leu	GRPr	—	596 (DMSO)	—	149	97
19	Arg-Arg-Arg-Arg	Cell-penetrating	386, 506, 540 (PBS)	654 (PBS)	0.13 (PBS)	58	54
20	Pro-Arg-Pro-Arg-Leu-Pro	Cell-penetrating	388, 506, 540(PBS)	652 (PBS)	0.08 (PBS)	69	54
21	Gly-Arg-Gly-Asp	Cancer cell	386, 507, 540(PBS)	654 (PBS)	0.13 (PBS)	47	54
22	Pro-Ala-Ala-Lys-Arg-Val-Lys-Lau-Asp	Nucleus	—	677 (dilute aerated aqueous solvent)	—	38	100
23	—	—	—	577 (PBS/CH3CN)	0.13 (PBS/CH3CN)	400	101
24	Tyr-Gly-Gly-Phe-Leu	Opiate receptors	266, 381, 416 (DMSO)	613 (DMSO)	0.021 (DMSO)	—	102
25	Phe-Phe-Lys	Self-assembly	—	600 (H2O)	—	—	105
26	Lys-urea-Glu	PMSA	—	670 (CH3CN)	0.0211 (CH3CN)	24, 375	106

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Therapeutic applications of iridium(III)–peptide bioconjugates

Historically, metal-based chemotherapeutic agents have mainly been platinum DNA-binding drugs; however, the use of these complexes has been associated with low targetability and severe side effects.^109,110 This has stimulated the development of other metallodrugs based on alternative metal centers such as iridium(III), as well as ruthenium(II), osmium(II), rhodium(II), as anticancer agents.^111,112 In 2016, López's group reported an iridium(III) complex modified with the positively charged octaarginine 27, which exhibits high-affinity sequence-selective DNA binding (Fig. 7).¹¹³ The positively charged peptide endows the iridium(III) complex with electrostatic affinity for negatively charged DNA. The binding affinity of the bioconjugate is dramatically increased compared with the parent peptide, and is also heavily dependent on the nuclearity of the bioconjugate. Bioconjugate 27 showed an increased luminescence at 620 nm with increasing concentrations of DNA hairpins upon excitation at 320 nm in PBS (100 mM) with NaCl (100 mM), pH 6.8. It also displayed higher cytotoxic activity against a series of tumor cell lines compared with the complex without octaarginine. Moreover bioconjugate 27 showed high cytotoxicity for doxorubicin-resistance NCI/ADR-RES cells, thus overcoming chemotherapeutic agent resistance. However, the detailed mechanism of action of this complex was not investigated.

Fig. 7 Chemical structures of bioconjugates 27–30.

Octaarginine also possesses CPP properties. In 2013, Keyes's group examined the conjugation of an iridium(III) luminophore to octaarginine (Fig. 7).¹¹⁴ The octaarginine peptide was prepared using a solid-phase peptide synthesizer. Unlike the parent complex, bioconjugate 28 was soluble in aqueous media and did not require pre-dissolution in an organic solvent. The UV–vis spectrum of bioconjugate 28 was dominated by a peptide absorbance peak at 262 nm an absorbance at 330 nm in PBS (pH 7.4). The MLCT absorbance peak at 387 nm was also apparent, whereas for the complex without the peptide, this band is broad, tailing to approximately 480 nm. The bioconjugate 28 exhibited a maximum emission at 543 nm in H₂O, which is slightly red-shifted compared with the parent (537 nm). Notably, bioconjugate 28 was rapidly and irreversibly transported across the cell membrane of both SP2 and CHO cells at room temperature. Moreover, bioconjugate 28 showed substantially higher cytotoxicity in both cell lines than the parent complex. The IC₅₀ values of 28 against SP2 and CHO cells were 34.95 μM and 54.44 μM, respectively, compared with 84.84 μM and 87.97 μM, respectively, for the parent iridium(III) complex. Overall, this work demonstrates the value of octaarginine conjugates for cell delivery of metal complexes.

Based on the iridium(III) coordination-based cRGD strategy, Fei's group also reported solvento iridium(III) complexes cyclized with oligoarginines as promoters of oncotic cell death (Fig. 7).¹¹⁵ The parent solvento complex or oligoarginine peptides alone showed no measurable toxicity (IC₅₀ > 100 μM). Encouragingly, the cytotoxicity of the iridium(III)–peptide bioconjugates against HeLa cells was distinctly enhanced upon iridium(III) coordination (IC₅₀ < 3.12 μM). Moreover, iridium(III) complexes with linear peptides were almost always less toxic than iridium(III) with cyclic peptide, especially for the peptides that have fewer numbers of arginines, possibly because of the higher hydrophobicity of linear peptide–iridium(III) complexes compared with their cyclic analogues. Cyclization of peptides could enhance their endocytosis, while the number of guanidine groups was related to their cell uptake efficiency. When the arginine number reached 8, both linear peptide–iridium(III) complexes and cyclic peptide–iridium(III) complexes showed similar cell uptake, suggesting that the flexibility of the linear structure may have been overwhelmed by a stronger electrostatic interaction between octaarginine and the cell membrane. The bioconjugates 29–30 with octaarginines were found to be most cytotoxic, and induced progressive oncotic cell death featuring cell membrane penetration and eruptive cytoplasmic content release. Experiments showed that the bioconjugate 30 utilized an energy-independent pathway to enter HeLa cells, where it distributed preferentially in the ER and the mitochondria rather than in the lysosomes. These bioconjugates can overcome multiple chemical drug resistances of cancer cells, and are immunogenic by stimulating dendritic cell maturation and inflammatory factor accumulation in mouse tumors.

In the targeted imaging studies, peptide conjugation of iridium(III) complexes lowered the cytotoxicity of the iridium(III)–peptide bioconjugates to normal cells, as shown by bioconjugates 18, 22 and 26. In a similar vein, targeted therapy is regarded as a promising strategy for overcoming the high cytotoxicity of metallodrugs due to poor selectivity against cancer cells.^89,116 In 2015, Aoki's group reported a triscyclometalated bioconjugate 31 containing a cationic peptide as an inducer and detector of cell death via a calcium-dependent pathway (Fig. 8).¹¹⁷ KKGG (Lys-Lys-Gly-Gly) is a cationic amphiphilic peptide with +9 net charge which selectively interacts with negatively charged cancer cell membranes owing to electrostatic and hydrophobic interactions. The amphiphilic triscyclometalated iridium(III) complex bioconjugate was designed by tethering a KKGG peptide, which is an inducer and detector of cell death, via an alkyl chain linker to the cyclometalated ligand. The bioconjugate 31 showed a maximum emission at 509 nm with a maximum excitation at 366 nm in degassed 100 mM HEPES (pH 7.4). Its absorption peaks were at 280 nm and 362 nm, while its emission lifetime was 1.7 μs. The bioconjugate showed lower toxicity against normal mouse lymphocytes compared with cancer cells, such as Jurkat and HeLa S3 cells, due to the presence of the KKGG peptide. In Jurkat cells, the bioconjugate interacts with anionic molecules on the surface and membrane receptors to trigger the Ca²⁺-dependent pathway and intracellular Ca²⁺ response, resulting in necrosis accompanied by membrane disruption. This work shows the potential of amphiphilic iridium(III)–peptide bioconjugates in anticancer drug discovery, but the role played by the iridium(III) complex part was not further clarified in this study. This work provides an excellent example of water-soluble peptide–iridium(III) complex conjugates for biological and biomedicinal applications. In 2016, López's group synthesized a new family of cyclometalated iridium(III) oligocationic peptides 32–33 conjugated with three arginine residues.¹¹⁸ The cyclic peptides bioconjugates 32–33 displayed IC₅₀ values in a similar range (19 and 21 μM, respectively) in NCI/ADR-RES cell lines, which are comparable to cisplatin (14 μM) (Fig. 8). Bioconjugates 32–33 localized and aggregated on the cell membrane, until they reached a threshold local concentration that allows them to behave like detergents and thus degrade the phospholipid bilayer causing cell death, which occurred on a much faster timescale than the toxicity effects of cisplatin.

Fig. 8 Chemical structures of bioconjugates 31–37.

Later on, Aoki's group further explored the Ca²⁺-related mechanism of this bioconjugate using proteomics, and introduced a photoaffinity unit, 3-trifluoromethyl-3-phenyldiazirine (TFPD), into at the end of the KKKGG peptide on the bioconjugate (Fig. 8).¹¹⁹ The protected TFPD-KKKGG peptide was prepared by Fmoc solid-phase peptide synthesis and was coupled with the amino group from the iridium(III) complex ligand, thereby producing bioconjugate 34 with +9 net charge. Bioconjugate 34 showed a maximum emission at 509 nm with a maximum excitation at 366 nm, and showed absorption peaks at 280 nm and 371 nm in degassed 100 mM HEPES. Three mechanisms were found: the first scenario involves the inhibition of (Ca²⁺-CaM)-PMCA complexation by iridium(III) complexes near the plasma membrane, thus preventing channel opening; the second involves the inhibition of (Ca²⁺-CaM)-K-Ras4B complexation by bioconjugate 34 to facilitate the phosphorylation of K-Ras4B in Jurkat cells; the third is the activation of the G-protein transduction pathway by the iridium(III) complexes, resulting in the release of Ca²⁺ from the ER to the cytoplasm in Jurkat cells. Overall, these mechanisms are associated with intracellular Ca²⁺ overload, thus inducing cell death.

In 2017, the same group further optimized the peptide substitution position with cationic peptides at the 4′ position of the 2-phenylpyridine ligand (Fig. 8).¹²⁰ The MTT experiment demonstrated that the 4′-KKGG ppy ligand had higher cytotoxicity compared with the 5′-KKGG ppy ligand in Jurkat cells. Moreover, the linker length is also key to their cytotoxicity; the eight-carbon alkyl length of bioconjugate 35 (EC₅₀ = 2.4 μM) on the 4′-ppy ligand had stronger cytotoxicity compared with other complexes with different linker lengths (29 μM for two-carbon alkyl length, 5.0 μM for six-carbon alkyl length, and 34 μM for sixteen-carbon alkyl length). These cytotoxicity results caused by linker length are consistent with previous studies, indicating that a balance between the hydrophobicity and hydrophilicity of the iridium(III) complex is an important factor. The bioconjugate 35 showed a maximum emission at 541 nm with a maximum excitation at 366 nm with a lifetime of 1.0 μs, and showed absorption peaks at 283 nm and 383 nm in degassed 100 mM HEPES. This work indicates that we need to carefully choose the connection position and linker length when designing iridium(III)–peptide bioconjugates. Later on, the group connected the KKKGG peptide through a nucleophilic substitution reaction on the 2-phenylpyridine ligand instead of through amide bond formation, to give the bioconjugate 36, with +12 net charge (Fig. 8).¹²¹ Bioconjugate 36 showed a maximum emission at 505 nm with a maximum excitation at 366 nm, with a lifetime of 0.78 μs. It also showed absorption peaks at 292 nm and 356 nm in degassed 100 mM HEPES. Bioconjugate 36 could induce paraptosis-like cell death of cancer cells via an intracellular Ca²⁺-dependent pathway in Jurkat cells with an EC₅₀ of 1.5 μM.

Aoki's group further introduced a hydrophobic N-heptadecanoyl group at the N-terminus of the KKKGG peptide to obtain the bioconjugate 37 (Fig. 8).¹²² The bioconjugate 37 showed a maximum emission at 501 nm with a maximum excitation at 365 nm, with a lifetime of 1.4 μs. Its absorption peaks were at 284 nm and 366 nm in degassed 100 mM HEPES. The MTT cytotoxicity assay showed an EC₅₀ of 1.5 μM against Jurkat cells, while 37 weakly induced cell death of IMR90 cells. It was found that bioconjugate 37 accumulated on the cell membrane and induced cell death via affecting the mitochondrial Ca²⁺-dependent pathway and an intracellular Ca²⁺ response. This study demonstrates that the appropriate choice of alkyl structure at the N-terminus in the peptide units could be used to tailor the lipophilicity of iridium(III)–peptide bioconjugates, which may enhance anticancer activity and improve cancer cell selectivity.

Necrosis and apoptosis are the two main pathways to cell death.^123,124 In 2018, Aoki's group designed and synthesized luminescent iridium(III)–peptide bioconjugate 38 for imaging cancer cells and inducing apoptosis (Fig. 9).¹²⁵ Bioconjugate 38 showed a maximum emission at 506 nm with a maximum excitation at 366 nm with a lifetime of 1000 ns, and showed absorption peaks at 286 nm and 360 nm in degassed DMSO at 25 °C. The hydrophilic SGSG sequence was inserted between the iridium(III) complex core and the cyclic peptide sequence WDCLDNRIGRRQCVKL as a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to improve the complex's solubility. Upon binding of TRAIL with death receptor 5 (DR5), this cyclic peptide sequence can interact with DR5 to induce cancer cytotoxicity dependent on the DR5 expression level of Jurkat cells. Bioconjugate 38 is the first example of an artificial luminescent TRAIL mimic that induces apoptosis-like cell death, although its cytotoxicity was only moderate.

Fig. 9 Chemical structures of bioconjugates 38–44.

Soon after, Aoki's group designed and synthesized another iridium(III)–peptide bioconjugate 39 similar to 38 to image cancer cells and induce necrosis-type cell death (Fig. 9).¹²⁶ Bioconjugate 39 showed a maximum emission at 506 nm with a maximum excitation at 366 nm with a lifetime of 1.3 μs, and showed absorption peaks at 285 nm and 361 nm in degassed DMSO. They found that cancer cell cytotoxicity of bioconjugate 39 was also dependent on the DR5 expression level of Jurkat cells, but bioconjugate 39 induced cell death via necrosis, as opposed to apoptosis for bioconjugate 38. The cell death induced by 39 was considered a necrosis-type cell death via a Ca²⁺-mediated intracellular signaling pathway.

In 2021, Aoki's group designed iridium(III) bioconjugates 40–42, containing three, two or one KKKGG sequences, respectively, to assess the effect of the number of peptide units on anticancer activity (Fig. 9).¹²⁷ Bioconjugates 40–42 exhibited a maximum emission at 507, 502 and 498 nm in degassed 100 mM HEPES and the emission quantum yields were determined to be 0.41, 0.44 and 0.12 in degassed 100 mM HEPES, respectively, and their lifetimes were determined to be 1.4, 1.6 and 1.8 μs, respectively. The EC₅₀ values of bioconjugates 40–42 against Jurkat cells were determined to be 1.5 μM, 4.1 μM, and 16.3 μM, respectively. These data show that there is a positive correlation between the number of H₂N-KKKGG peptide groups and the degree of cytotoxicity against Jurkat cells. Meanwhile, the cytotoxicity of bioconjugates 40–42 was relatively lower against other cell lines, including cancer cells HeLa S3 (EC₅₀ > 50 μM) and A549 cells (EC₅₀ > 39.3 μM), and human diploid cells IMR90 cells (EC₅₀ > 18.4 μM). Recently, the same group discovered that these bioconjugates could induce cell death through new pathways: (1) by triggering paraptosis in cancer cells via an intracellular Ca²⁺ overload from the ER and a decrease in mitochondrial membrane potential,¹²⁸ and (2) mitochondrial Ca²⁺ overload triggered by membrane fusion between mitochondria and the ER.¹²⁹

Click chemistry is a versatile methodology for establishing structurally diverse iridium(III)–peptide bioconjugates. Cu(I)-catalyzed azide–alkyne cycloadditions have been a benchmark in many areas of recent synthetic chemistry; however, side reactions and toxicity of the copper catalyst often limits its utilization in biological applications. By replacing terminal alkynes with strain-promoted cycloalkynes, copper-free azide–alkyne cycloadditions can be achieved under mild conditions without disrupting the function of the biomolecules. In 2019, Sadler's group reported a strategy for conjugating an iridium(III) complex to target peptides via copper-free click chemistry.¹³⁰ They synthesized a half-sandwich Cp* iridium(III) complex with a dibenzocyclooctyne moiety 43, which showed moderate cytotoxicity against human ovarian cancer cells A2780. The center of the iridium(III) complex can catalyze hydride transfer from NADH to molecular oxygen, generating hydrogen peroxide in cancer cells to trigger cell death. However, it lacked selectivity against tumor cells over normal cells. Subsequently, complex 43 was conjugated with a tumor-targeting cyclic nonapeptide c(CRWYDENAC) to afford bioconjugate 44 (Fig. 9). Bioconjugate 44 had enhanced anticancer activity and selectivity due to the introduction of the peptide (Table 3).

Table 3 Photophysical properties of bioconjugates 27–44

Bioconjugate	Peptide	Target	λ abs/nm	λ emi/nm	Φ PL	Lifetime/ns	Ref.
27	Octa-arginine	DNA	—	—	—	—	113
28	Octa-arginine	Cell-penetrating	262, 330, 387(PBS)	543 (H2O)	—	—	114
29	Octa-arginine	Cell-penetrating	—	—	—	—	115
30	Octa-arginine	Cell-penetrating	—	—	—	—	115
31	Lys-Lys-Gly-Gly	Cell surface/membrane receptors	280, 362 (HEPES)	509 (HEPES)	0.55 (0.1 M H2SO4)	1700	117
32	Arg-Arg-Arg		—	—	—	—	118
33	Arg-Arg-Arg		—	—	—	—	118
34	Lys-Lys-Lys-Gly-Gly	Cell surface/membrane receptors	280, 371 (HEPES)	498, 509 (HEPES)	0.0024/0.0062 (0.1 M H2SO4)		119
35	Lys-Lys-Gly-Gly	Cell surface/membrane receptors	283, 383 (HEPES)	541 (HEPES)	0.11 (0.1 M H2SO4)	1000	120
36	Lys-Lys-Lys-Gly-Gly	Cell surface/membrane receptors	292, 356 (HEPES)	505 (HEPES)	0.43 (0.1 M H2SO4)	780	121
37	Lys-Lys-Lys-Gly-Gly	Cell surface/membrane receptors	284, 366 (HEPES)	501 (HEPES)	0.52 (0.1 M H2SO4)	1400	122
38	DR-binding peptide	DR5	286, 360 (degassed DMSO)	506 (degassed DMSO)	0.35 (0.1 M H2SO4)	1000	125
39	DR-binding peptide	DR5	285, 361 (degassed DMSO)	506 (degassed DMSO)	0.33 (0.1 M H2SO4)	1300	126
40	Lys-Lys-Lys-Gly-Gly	Cell surface/membrane receptors	280, 360 (degassed HEPES)	507 (degassed HEPES)	0.50 (0.1 M H2SO4)	1400	127
41	Lys-Lys-Lys-Gly-Gly	Cell surface/membrane receptors	277, 358 (degassed HEPES)	502 (degassed HEPES)	0.41 (0.1 M H2SO4)	1600	127
42	Lys-Lys-Lys-Gly-Gly	Cell surface/membrane receptors	278, 352 (degassed HEPES)	498 (degassed HEPES)	0.12 (0.1 M H2SO4)	1800	127
43	—	—	—	—	—	—	130
44	c(Cys-Arg-Typ-Tyr-Asp-Glu-Asn-Ala-Cys)	Integrin α6 receptor	—	—	—	—	130

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Conclusion and prospects

In this work, we have briefly introduced the developing history of iridium(III)–peptide bioconjugates from the inspiration of the interaction between iridium(III) complexes and Aβ peptides and advances in peptide-based targeted drug delivery. We summarized initial efforts on coordination of solvent iridium(III) complex to histidine and histidine-derived peptides, and later iridium(III)–peptide bioconjugates were prepared using traditional amide bond formation strategies. We further surveyed the imaging and therapeutic applications of iridium(III)–peptide bioconjugates. We noted that click chemistry and bimetallic strategies have been applied for developing structurally diverse iridium(III)–peptide bioconjugates, but only a few examples are currently available. There is a large space for harnessing other types of click reaction for establishing iridium(III)–peptide bioconjugates, such as inverse electron-demand Diels–Alder (iEDDA) cycloaddition and photoinitiated reactions, and exploring the combination of iridium(III) complexes with other metal centers, such as platinum(II), rhodium(III), osmium(II) and ruthenium(II), is also worth exploring. In addition, imaging studies of iridium(III)–peptide bioconjugates are almost all confined to in cellulo experiments, largely because available bioconjugates are limited by short-wavelength emission and weak absorption over 500 nm. Only our group has applied an iridium(III)–peptide bioconjugate for initial in vivo evaluations. Thus, there is significant room for the exploration of in vivo applications of iridium(III)–peptide bioconjugates, which is required for further clinical translation. Furthermore, although a variety of peptides are used in the field of PDCs, such as cell-penetrating peptides (linear and cyclic cell-penetrating peptides), cell-targeting peptides (RGD peptide, gonadotropin-releasing hormone peptides, somatostatin mimetic peptides), self-assembling peptides, and responsive peptides (temperature, pH, enzymes), the peptides used in iridium(III)–peptide bioconjugates are very limited.⁴⁵

In the future, there are some directions for accelerating the clinical transition of iridium(III)–peptide bioconjugates. NIR iridium(III)–peptide bioconjugates with stronger absorption and emission over 500 nm will be preferred due to their better tissue penetration. The cyclometalated ligand plays an important role in modulating emission and absorption properties. 2-Phenylquinoxaline has been demonstrated to be a relatively simple cyclometalated ligand for enabling NIR emission in iridium(III) complexes,¹³¹ while fluorophores are also good choices as ligands.¹³² Photoacoustic and ultrasound-induced luminescence abilities of iridium(III)–peptide bioconjugates could also be investigated, as these techniques have better penetration in vivo.^133,134 The in vivo therapeutic potential of iridium(III)–peptide bioconjugates must be systematically evaluated, while their anticancer mechanisms also need to be clarified. In addition, the therapeutic application of iridium(III)–peptide bioconjugates in other diseases apart from cancer is often overlooked, but is worth exploring due to the known abilities of iridium(III) complexes to reduce liver injury and promote diabetic wound healing, among other applications.^135,136 Furthermore, based on the advances in the PDCs, the development of new G protein-coupled receptor-binding peptides would be also desirable to advance iridium–peptide conjugates to the level of ADCs. With further investment, we believe that iridium(III)–peptide bioconjugates can make a significant impact in biomedical science, similar to the success of other conjugate strategies such as ADC and PROTAC.

Author contributions

Conceptualization, W. W., C.-H. L. and J. W.; methodology, W. W. and S. J.; data curation, S. J., X. W. and J. D.; writing – original draft preparation, S. J. and X. W.; writing – review and editing, W. W., C.-H. L., J. W., S.-C. N., J. D., D. S.-H. C., and C.-Y. W.; supervision, W. W., C.-H. L., J. W. and C.-Y. W.; project administration, W. W., C.-H. L. and J. W.; funding acquisition, W. W., C.-H. L. and J. W. All authors have read and agreed to the published version of the manuscript.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work is supported by the National Natural Science Foundation of China (22101230), the Fundamental Research Funds for the Central Universities (D5000230060), the Key Research and Development Program of Shaanxi (2024SF-YBXM-181, 2024SF-YBXM-418), Shanghai Sailing Program (21YF1451200), the Natural Science Foundation of Chongqing, China (cstc2021jcyj-msxm2073), the Shaanxi Fundamental Science Research Project for Chemistry & Biology (22JHQ082), the Guangdong Basic and Applied Basic Research Foundation (2021A1515110840, 2023A1515011871), the Science and Technology Development Fund, Macau SAR, China (File no. 005/2023/SKL, 0020/2022/A1, 0045/2023/AMJ, 0032/2023/RIB2), the University of Macau, Macau SAR, China (File no. MYRG2020-00017-ICMS, MYRG2022-00137-ICMS, MYRG-GRG2023-00194-ICMS-UMDF), the State Key Laboratory of Quality Research in Chinese Medicine, the University of Macau, Macau SAR, China (File no. SKL-QRCM-IRG2023-025).

References

1. Q. Zhang and K. M.-C. Wong, Photophysical, ion-sensing and biological properties of rhodamine-containing transition metal complexes, Coord. Chem. Rev., 2020, 416, 213336.
2. I. B. Sivaev and V. I. Bregadze, 1,1′-Bis(ortho-carborane)-based transition metal complexes, Coord. Chem. Rev., 2019, 392, 146–176.
3. H. Na and T. S. Teets, Highly luminescent cyclometalated iridium complexes generated by nucleophilic addition to coordinated isocyanides, J. Am. Chem. Soc., 2018, 140, 6353–6360.
4. Q. Zhao, C. Huang and F. Li, Phosphorescent heavy-metal complexes for bioimaging, Chem. Soc. Rev., 2011, 40, 2508–2524.
5. J. Shen, T. W. Rees, L. Ji and H. Chao, Recent advances in ruthenium(II) and iridium(III) complexes containing nanosystems for cancer treatment and bioimaging, Coord. Chem. Rev., 2021, 443, 214016.
6. S. Monro, K. L. Colón, H. Yin, J. Roque III, P. Konda, S. Gujar, R. P. Thummel, L. Lilge, C. G. Cameron and S. A. McFarland, Transition metal complexes and photodynamic therapy from a tumor-centered approach: Challenges, opportunities, and highlights from the development of TLD1433, Chem. Rev., 2019, 119, 797–828.
7. K. K.-W. Lo, Luminescent rhenium(I) and iridium(III) polypyridine complexes as biological probes, imaging reagents, and photocytotoxic agents, Acc. Chem. Res., 2015, 48, 2985–2995.
8. F. Heinemann, J. Karges and G. Gasser, Critical overview of the use of Ru(II) polypyridyl complexes as photosensitizers in one-photon and two-photon photodynamic therapy, Acc. Chem. Res., 2017, 50, 2727–2736.
9. K. K.-W. Lo, M.-W. Louie and K. Y. Zhang, Design of luminescent iridium(III) and rhenium(I) polypyridine complexes as in vitro and in vivo ion, molecular and biological probes, Coord. Chem. Rev., 2010, 254, 2603–2622.
10. L. M. Lifshits, J. A. Roque III, H. D. Cole, R. P. Thummel, C. G. Cameron and S. A. McFarland, NIR-absorbing Ru(II) complexes containing α-oligothiophenes for applications in photodynamic therapy, ChemBioChem, 2020, 21, 3594–3607.
11. G. Li, Q. Lin, L. Sun, C. Feng, P. Zhang, B. Yu, Y. Chen, Y. Wen, H. Wang, L. Ji and H. Chao, A mitochondrial targeted two-photon iridium(III) phosphorescent probe for selective detection of hypochlorite in live cells and in vivo, Biomaterials, 2015, 53, 285–295.
12. H. Huang, P. Zhang, B. Yu, Y. Chen, J. Wang, L. Ji and H. Chao, Targeting nucleus DNA with a cyclometalated dipyridophenazine ruthenium(II) complex, J. Med. Chem., 2014, 57, 8971–8983.
13. X. Li, N. Kwon, T. Guo, Z. Liu and J. Yoon, Innovative strategies for hypoxic-tumor photodynamic therapy, J. Mater. Chem. B, 2018, 57, 11522–11531.
14. T. Yoshihara, S. Murayama, T. Masuda, T. Kikuchi, K. Yoshida, M. Hosaka and S. Tobita, Mitochondria-targeted oxygen probes based on cationic iridium complexes with a 5-amino-1, 10-phenanthroline ligand, J. Photochem. Photobiol., A, 2015, 299, 172–182.
15. J. Shen, J. Karges, K. Xiong, Y. Chen, L. Ji and H. Chao, Cancer cell membrane camouflaged iridium complexes functionalized black-titanium nanoparticles for hierarchical-targeted synergistic NIR-II photothermal and sonodynamic therapy, Biomaterials, 2021, 275, 120979.
16. Y. Hu, R. Lin, K. Patel, A. G. Cheetham, C. Kan and H. Cui, Spatiotemporal control of the creation and immolation of peptide assemblies, Coord. Chem. Rev., 2016, 320–321, 2–17.
17. P. G. Yap and C. Y. Gan, In vivo challenges of anti-diabetic peptide therapeutics: Gastrointestinal stability, toxicity and allergenicity, Trends Food Sci. Technol., 2020, 105, 161–175.
18. R. S. Tu and M. Tirrell, Bottom-up design of biomimetic assemblies, Adv. Drug Delivery Rev., 2004, 56, 1537–1563.
19. F. Zhao, M. L. Ma and B. Xu, Molecular hydrogels of therapeutic agents, Chem. Soc. Rev., 2009, 38, 883–891.
20. J. Boekhoven and S. I. Stupp, 25th Anniversary article: Supramolecular materials for regenerative medicine, Adv. Mater., 2014, 26, 1642–1659.
21. J. Wang, X. Wang, K. Yang, S. Hu and W. Wang, Self-assembly of small organic molecules into luminophores for cancer theranostic applications, Biosensors, 2022, 12, 683.
22. R. J. Boohaker, M. W. Lee, P. Vishnubhotla, J. M. Perez and A. R. Khaled, The use of therapeutic peptides to target and to kill cancer cells, Curr. Med. Chem., 2012, 19, 3794–3804.
23. S. Marqus, E. Pirogova and T. J. Piva, Evaluation of the use of therapeutic peptides for cancer treatment, J. Biomed. Sci., 2017, 24, 21–35.
24. J. M. Shin, J. W. Gwak, P. Kamarajan, J. C. Fenno, A. H. Rickard and Y. L. Kapila, Biomedical applications of nisin, J. Appl. Microbiol., 2016, 120, 1449–1465.
25. M. C. Bagley, J. W. Dale, E. A. Merritt and X. Xiong, Thiopeptide antibiotics, Chem. Rev., 2005, 105, 685–714.
26. M. Muttenthaler, G. F. King, D. J. Adams and P. F. Alewood, Trends in peptide drug discovery, Nat. Rev. Drug Discovery, 2021, 20, 309–325.
27. S.-H. Wang and J. Yu, Structure-based design for binding peptides in anti-cancer therapy, Biomaterials, 2018, 156, 1–15.
28. T. Bruckdorfer, O. Marder and F. Albericio, From production of peptides in milligram amounts for research to multi-tons auantities for drugs of the future, Curr. Pharm. Biotechnol., 2004, 5, 29–43.
29. A. A. Zompra, A. S. Galanis, O. Werbitzky and F. Albericio, Manufacturing peptides as active pharmaceutical ingredients, Future Med. Chem., 2009, 1, 361–377.
30. P. Vlieghe, V. Lisowski, J. Martinez and M. Khrestchatisky, Synthetic therapeutic peptides: science and market, Drug Discovery Today, 2010, 15, 40–56.
31. D. J. Craik, D. P. Fairlie, S. Liras and D. Price, The future of peptide-based drugs, Chem. Biol. Drug Des., 2013, 81, 136–147.
32. B. Kar, N. Roy, S. Pete, P. Moharana and P. Paira, Ruthenium and iridium based mononuclear and multinuclear complexes: A breakthrough of next-generation anticancer metallopharmaceuticals, Inorg. Chim. Acta, 2020, 512, 119858.
33. G. Gupta, A. Das, J. Lee, N. Mandal and C. Y. Lee, Multinuclear Ir-BODIPY complexes: Synthesis and binding studies, Inorg. Chem. Commun., 2020, 113, 107759.
34. S. Lamansky, P. Djurovich, D. Murphy, F. Abdel-Razzaq, H.-E. Lee, C. Adachi, P. E. Burrows, S. R. Forrest and M. E. Thompson, Highly phosphorescent bis-cyclometalated iridium complexes: Synthesis, photophysical characterization, and use in organic light emitting diodes, J. Am. Chem. Soc., 2001, 123, 4304–4312.
35. F. Monti, A. Baschieri, L. Sambri and N. Armaroli, Excited-state engineering in heteroleptic ionic iridium(III) complexes, Acc. Chem. Res., 2021, 54, 1492–1505.
36. G. Gupta, P. Kumari, J. Y. Ryu, J. Lee, S. M. Mobin and C. Y. Lee, Mitochondrial localization of highly fluorescent and photostable BODIPY-based ruthenium(II), rhodium(III), and iridium(III) metal complexes, Inorg. Chem., 2019, 58, 8587–8595.
37. K. K.-W. Lo, Molecular design of bioorthogonal probes and imaging reagents derived from photofunctional transition metal complexes, Acc. Chem. Res., 2020, 53, 32–44.
38. L. C.-C. Lee and K. K.-W. Lo, Strategic design of luminescent rhenium(I), ruthenium(II), and iridium(III) complexes as activity-based probes for bioimaging and biosensing, Chem. – Asian J., 2022, 17, e202200840.
39. L. Ke, F. Wei, L. Xie, J. Karges, Y. Chen, L. Ji and H. Chao, A biodegradable iridium(III) coordination polymer for enhanced two-photon photodynamic therapy using an apoptosis–ferroptosis hybrid pathway, Angew. Chem., Int. Ed., 2022, 61, e202205429.
40. A. Pinto, U. Hoffmanns, M. Ott, G. Fricker and N. Metzler-Nolte, Modification with organometallic compounds improves crossing of the blood–brain barrier of [Leu5]-enkephalin derivatives in an in vitro model system, ChemBioChem, 2009, 10, 1852–1860.
41. J. C. Reubi, Peptide receptors as molecular targets for cancer diagnosis and therapy, Endocr. Rev., 2003, 24, 389–427.
42. S. M. P. Vadevoo, S. Gurung, H.-S. Lee, G. R. Gunassekaran, S.-M. Lee, J.-W. Yoon, Y.-K. Lee and B. Lee, Peptides as multifunctional players in cancer therapy, Exp. Mol. Med., 2023, 55, 1099–1109.
43. Y. Han, Z. Gao, L. Chen, L. Kang, W. Huang, M. Jin, Q. Wang and Y. H. Bae, Multifunctional oral delivery systems for enhanced bioavailability of therapeutic peptides/proteins, Acta Pharm. Sin. B, 2019, 9, 902–922.
44. L. C.-C. Lee, L. Huang, P. K.-K. Leung and K. K.-W. Lo, Recent development of photofunctional transition metal–peptide conjugates for bioimaging and therapeutic applications, Eur. J. Inorg. Chem., 2022, 2022, e202200455.
45. B. M. Cooper, J. Iegre, D. H. O’Donovan, M. Ö. Halvarsson and D. R. Spring, Peptides as a platform for targeted therapeutics for cancer: peptide–drug conjugates (PDCs), Chem. Soc. Rev., 2021, 50, 1480–1494.
46. A. F. B. Räder, F. Reichart, M. Weinmüller and H. Kessler, Improving oral bioavailability of cyclic peptides by N-methylation, Bioorg. Med. Chem., 2018, 26, 2766–2773.
47. L. Gong, H. Zhao, Y. Liu, H. Wu, C. Liu, S. Chang, L. Chen, M. Jin, Q. Wang, Z. Gao and W. Huang, Research advances in peptide-drug conjugates, Acta Pharm. Sin. B, 2023, 13, 3659–3677.
48. C. Fu, L. Yu, Y. Miao, X. Liu, Z. Yu and M. Wei, Peptide–drug conjugates (PDCs): A novel trend of research and development on targeted therapy, hype or hope?, Acta Pharm. Sin. B, 2023, 13, 498–516.
49. X. Ji, A. L. Nielsen and C. Heinis, Cyclic peptides for drug development, Angew. Chem., Int. Ed., 2024, 63, e202308251.
50. V. Adebomi, R. D. Cohen, R. Wills, H. A. H. Chavers, G. E. Martin and M. Raj, CyClick Chemistry for the Synthesis of Cyclic Peptides, Angew. Chem., Int. Ed., 2019, 58, 19073–19080.
51. S. M. Meier-Menches and A. Casini, Design strategies and medicinal applications of metal-peptidic bioconjugates, Bioconjugate Chem., 2020, 31, 1279–1288.
52. M. Salmain, N. Fischer-Durand and B. Rudolf, Bioorthogonal conjugation of transition organometallic complexes to peptides and proteins: Strategies and applications, Eur. J. Inorg. Chem., 2020, 2020, 21–35.
53. Q. Y. Hu, F. Berti and R. Adamo, Towards the next generation of biomedicines by site-selective conjugation, Chem. Soc. Rev., 2016, 45, 1691–1719.
54. K. Koren, R. I. Dmitriev, S. M. Borisov, D. B. Papkovsky and I. Klimant, Complexes of Ir(III)-octaethylporphyrin with peptides as probes for sensing cellular O₂, ChemBioChem, 2012, 13, 1184–1190.
55. J. Kuil, P. Steunenberg, P. T. K. Chin, J. Oldenburg, K. Jalink, A. H. Velders and F. W. B. van Leeuwen, Peptide-functionalized luminescent iridium complexes for lifetime imaging of CXCR4 expression, ChemBioChem, 2011, 12, 1897–1903.
56. C. A. Wootton, A. J. Millett, A. F. Lopez-Clavijo, C. K. C. Chiu, M. P. Barrow, G. J. Clarkson, P. J. Sadler and P. B. O'Connor, Structural analysis of peptides modified with organo-iridium complexes, opportunities from multi-mode fragmentation, Analyst, 2019, 144, 1575–1581.
57. B. Albada and N. Metzler-Nolte, Organometallic–peptide bioconjugates: synthetic strategies and medicinal applications, Chem. Rev., 2016, 116, 11797–11839.
58. M. Martínez-Alonso and G. Gasser, Ruthenium polypyridyl complex-containing bioconjugates, Coord. Chem. Rev., 2021, 434, 213736.
59. A. Monney and M. Albrecht, Transition metal bioconjugates with an organometallic link between the metal and the biomolecular scaffold, Coord. Chem. Rev., 2013, 257, 2420–2433.
60. C. Bacchella, S. Dell'Acqua, S. Nicolis, E. Monzani and L. Casella, The reactivity of copper complexes with neuronal peptides promoted by catecholamines and its impact on neurodegeneration, Coord. Chem. Rev., 2022, 471, 214756.
61. K. S. Gkika, D. Cullinane and T. E. Keyes, Metal peptide conjugates in cell and tissue imaging and biosensing, Top. Curr. Chem., 2022, 380, 30.
62. T. Yoshihara, Y. Yamaguchi, M. Hosaka, T. Takeuchi and S. Tobita, Ratiometric molecular sensor for monitoring oxygen levels in living cells, Angew. Chem., Int. Ed., 2012, 51, 4148–4151.
63. M. Alas, A. Saghaeidehkordi and K. Kaur, Peptide–drug conjugates with different linkers for cancer therapy, J. Med. Chem., 2021, 64, 216–232.
64. R. Ren, J. Qi, S. Lin, X. Liu, P. Yin, Z. Wang, R. Tang, J. Wang, Q. Huang, J. Li, X. Xie, Y. Hu, S. Cui, Y. Zhu, X. Yu, P. Wang, Y. Zhu, Y. Wang, Y. Huang, Y. Hu, Y. Wang, C. Li, M. Zhou and G. Wang, The China alzheimer report 2022, Gen. Psychiatry, 2022, 35, e100751.
65. T. S. Chisholm and C. A. Hunter, A closer look at amyloid ligands, and what they tell us about protein aggregates, Chem. Soc. Rev., 2024, 53, 1354–1374.
66. B. Feng, X. Lu, G. Zhang, L. Zhao and D. Mei, STING agonist delivery by lipid calcium phosphate nanoparticles enhances immune activation for neuroblastoma, Acta Mater. Med., 2023, 2, 216–227.
67. P. Faller and C. Hureau, Bioinorganic chemistry of copper and zinc ions coordinated to amyloid-β peptide, Dalton Trans., 2009, 1080–1094.
68. K. P. Kepp and R. Squitti, Copper imbalance in Alzheimer's disease: Convergence of the chemistry and the clinic, Coord. Chem. Rev., 2019, 397, 168–187.
69. D.-L. Ma, W.-L. Wong, W.-H. Chung, F.-Y. Chan, P.-K. So, T.-S. Lai, Z.-Y. Zhou, Y.-C. Leung and K.-Y. Wong, A highly selective luminescent switch-on probe for histidine/histidine-rich proteins and its application in protein staining, Angew. Chem., Int. Ed., 2008, 47, 3735–3739.
70. B. Y.-W. Man, H.-M. Chan, C.-H. Leung, D. S.-H. Chan, L.-P. Bai, Z.-H. Jiang, H.-W. Li and D.-L. Ma, Group 9 metal-based inhibitors of β-amyloid (1–40) fibrillation as potential therapeutic agents for Alzheimer's disease, Chem. Sci., 2011, 2, 917–921.
71. J.-M. Suh, G. Kim, J. Kang and M. H. Lim, Strategies employing transition metal complexes to modulate amyloid-β aggregation, Inorg. Chem., 2019, 58, 8–17.
72. L. Lu, H.-J. Zhong, M. Wang, S.-L. Ho, H.-W. Li, C.-H. Leung and D.-L. Ma, Inhibition of beta-amyloid fibrillation by luminescent iridium(III) complex probes, Sci. Rep., 2015, 5, 14619.
73. J. Karges, Clinical development of metal complexes as photosensitizers for photodynamic therapy of cancer, Angew. Chem., Int. Ed., 2022, 61, e202112236.
74. P. Tao, Z. Lv, X.-K. Zheng, H. Jiang, S. Liu, H. Wang, W.-Y. Wong and Q. Zhao, Isomer engineering of lepidine-based iridophosphors for far-red hypoxia imaging and photodynamic therapy, Inorg. Chem., 2022, 61, 17703–17712.
75. Y. Wu, S. Li, Y. Chen, W. He and Z. Guo, Recent advances in noble metal complex based photodynamic therapy, Chem. Sci., 2022, 13, 5085–5106.
76. X. Zhao, J. Liu, J. Fan, H. Chao and X. Peng, Recent progress in photosensitizers for overcoming the challenges of photodynamic therapy: from molecular design to application, Chem. Soc. Rev., 2021, 50, 4185–4219.
77. V. Novohradsky, A. Rovira, C. Hally, A. Galindo, G. Vigueras, A. Gandioso, M. Svitelova, R. Bresolí-Obach, H. Kostrhunova, L. Markova, J. Kasparkova, S. Nonell, J. Ruiz, V. Brabec and V. Marchán, Towards novel photodynamic anticancer agents generating superoxide anion radicals: A cyclometalated Ir^III complex conjugated to a far-red emitting coumarin, Angew. Chem., Int. Ed., 2019, 58, 6311–6315.
78. A. Rovira, E. Ortega-Forte, C. Hally, M. Jordà-Redondo, D. Abad-Montero, G. Vigueras, J. I. Martínez, M. Bosch, S. Nonell, J. Ruiz and V. Marchán, Exploring structure–activity relationships in photodynamic therapy anticancer agents based on Ir(III)-COUPY conjugates, J. Med. Chem., 2023, 66, 7849–7867.
79. J. Kang, S. J. C. Lee, J. S. Nam, H. J. Lee, M.-G. Kang, K. J. Korshavn, H.-T. Kim, J. Cho, A. Ramamoorthy, H.-W. Rhee, T.-H. Kwon and M. H. Lim, An iridium(III) complex as a photoactivatable tool for oxidation of amyloidogenic peptides with subsequent modulation of peptide aggregation, Chem. – Eur. J., 2017, 23, 1645–1653.
80. J. Kang, J. S. Nam, H. J. Lee, G. Nam, H.-W. Rhee, T.-H. Kwon and M. H. Lim, Chemical strategies to modify amyloidogenic peptides by iridium(III) complexes: coordination and photo-induced oxidation, Chem. Sci., 2019, 10, 6855–6862.
81. M. Kim, G. Gupta, J. Lee, C. Na, J. Kwak, Y. Lin, Y.-H. Lee, M. H. Lim and C. Y. Lee, Metal–BODIPY complexes: versatile photosensitizers for oxidizing amyloid-β peptides and modulating their aggregation profiles, Inorg. Chem. Front., 2024, 11, 1966–1977.
82. Q. Jiang, M. Wang, L. Yang, H. Chen and L. Mao, Synergistic coordination and hydrogen bonding interaction modulate the emission of iridium complex for highly sensitive glutamine imaging in live cells, Anal. Chem., 2016, 88, 10322–10327.
83. A. D. de Bruijn and G. Roelfes, Chemical modification of dehydrated amino acids in natural antimicrobial peptides by photoredox catalysis, Chem. – Eur. J., 2018, 24, 11314–11318.
84. R. C. W. v. Lier, A. D. d. Bruijn and G. Roelfes, A water-soluble iridium photocatalyst for chemical modification of dehydroalanines in peptides and proteins, Chem. – Eur. J., 2020, 27, 1430–1437.
85. X. Wang, J. Jia, Z. Huang, M. Zhou and H. Fei, Luminescent peptide labeling based on a histidine-binding iridium(III) complex for cell penetration and intracellular targeting studies, Chem. – Eur. J., 2011, 17, 8028–8032.
86. X. Ma, J. Jia, R. Cao, X. Wang and H. Fei, Histidine-iridium(III) coordination-based peptide luminogenic cyclization and cyclo-RGD peptides for cancer-cell targeting, J. Am. Chem. Soc., 2014, 136, 17734–17737.
87. J. Kuil, P. Steunenberg, P. T. K. Chin, J. Oldenburg, K. Jalink, A. H. Velders and F. W. B. v. Leeuwen, Peptide-functionalized luminescent iridium complexes for lifetime imaging of CXCR4 expression, ChemBioChem, 2011, 12, 1897–1903.
88. F. Balkwill, The significance of cancer cell expression of the chemokine receptor CXCR4, Semin. Cancer Biol., 2004, 14, 171–179.
89. R. Paprocka, M. Wiese-Szadkowska, S. Janciauskiene, T. Kosmalski, M. Kulik and A. Helmin-Basa, Latest developments in metal complexes as anticancer agents, Coord. Chem. Rev., 2022, 452, 214307.
90. I. S. Kritchenkov, D. D. Zhukovsky, A. Mohamed, V. A. Korzhikov-Vlakh, T. B. Tennikova, A. Lavrentieva, T. Scheper, V. V. Pavlovskiy, V. V. Porsev, R. A. Evarestov and S. P. Tunik, Functionalized Pt(II) and Ir(III) NIR emitters and their covalent conjugates with polymer-based nanocarriers, Bioconjugate Chem., 2020, 31, 1327–1343.
91. G. Lavarda, D. Shimizu, T. Torres and A. Osuka, meso-(2-Pyridyl)-boron(III)-subporphyrin: Perimeter iridium(III) coordination, Angew. Chem., Int. Ed., 2020, 59, 3127–3130.
92. C. Huang, C. Liang, T. Sadhukhan, S. Banerjee, Z. Fan, T. Li, Z. Zhu, P. Zhang, K. Raghavachari and H. Huang, In vitro and in vivo photocatalytic cancer therapy with biocompatible iridium(III) photocatalysts, Angew. Chem., Int. Ed., 2021, 60, 9474–9479.
93. T. M. Stonelake, K. A. Phillips, H. Y. Otaif, Z. C. Edwardson, P. N. Horton, S. J. Coles, J. M. Beames and S. J. A. Pope, Spectroscopic and theoretical investigation of color tuning in deep-red luminescent iridium(III) complexes, Inorg. Chem., 2020, 59, 2266–2277.
94. K. Vellaisamy, G. Li, W. Wang, C.-H. Leung and D.-L. Ma, A long-lived peptide-conjugated iridium(III) complex as a luminescent probe and inhibitor of the cell migration mediator, formyl peptide receptor 2, Chem. Sci., 2018, 9, 8171–8177.
95. N. Prevete, F. Liotti, G. Marone, R. M. Melillo and A. de Paulis, Formyl peptide receptors at the interface of inflammation, angiogenesis and tumor growth, Pharmacol. Res., 2015, 102, 184–191.
96. V. Novohradsky, A. Zamora, A. Gandioso, V. Brabec, J. Ruiz and V. Marchán, Somatostatin receptor-targeted organometallic iridium(III) complexes as novel theranostic agents, Chem. Commun., 2017, 53, 5523–5526.
97. W. Wang, K.-J. Wu, K. Vellaisamy, C.-H. Leung and D.-L. Ma, Peptide-conjugated long-lived theranostic imaging for targeting GRPr in cancer and immune cells, Angew. Chem., Int. Ed., 2020, 59, 17897–17902.
98. G. Fang, D. Liu, M. Zhang, L. Shao, X. Shao, J. Chen, C. Meng, Y. Wang, K. Zeng and Q. Chen, Single-organelle localization-based super-resolution imaging for subcellular molecules micro-dynamics, Coord. Chem. Rev., 2024, 504, 215670.
99. S. Shaikh, Y. Wang, F. u. Rehman, H. Jiang and X. Wang, Phosphorescent Ir(III) complexes as cellular staining agents for biomedical molecular imaging, Coord. Chem. Rev., 2020, 416, 213344.
100. A. H. Day, M. H. Übler, H. L. Best, E. Lloyd-Evans, R. J. Mart, I. A. Fallis, R. K. Allemann, E. A. H. Al-Wattar, N. I. Keymer, N. J. Buurma and S. J. A. Pope, Targeted cell imaging properties of a deep red luminescent iridium(III) complex conjugated with a c-Myc signal peptide., Chem. Sci., 2020, 11, 1599–1606.
101. L. C.-C. Lee, A. W.-Y. Tsang, H.-W. Liu and K. K.-W. Lo, Photofunctional cyclometalated iridium(III) polypyridine complexes bearing a perfluorobiphenyl moiety for bioconjugation, bioimaging, and phototherapeutic applications, Inorg. Chem., 2020, 59, 14796–14806.
102. A. Luengo, I. Marzo, M. Reback, I. M. Daubit, V. Fernández-Moreira, N. Metzler-Nolte and M. C. Gimeno, Luminescent bimetallic Ir(III)/Au(I) peptide bioconjugates as potential theranostic agents., Chem. – Eur. J., 2020, 26, 12158–12167.
103. K. A. Roth and J. D. Barchas, Small cell carcinoma cell lines contain opioid peptides and receptors, Cancer, 1986, 57, 769–773.
104. Z. Liu, J. Guo, Y. Qiao and B. Xu, Enzyme-instructed intracellular peptide assemblies, Acc. Chem. Res., 2023, 56, 3076–3088.
105. C. Jin, G. Li, X. Wu, J. Liu, W. Wu, Y. Chen, T. Sasaki, H. Chao and Y. Zhang, Robust packing of a self-assembling iridium complex via endocytic trafficking for long-term lysosome tracking, Angew. Chem., Int. Ed., 2021, 60, 7597–7601.
106. X. Yang, S.-C. Nao, C. Lin, L. Kong, J. Wang, C.-N. Ko, J. Liu, D.-L. Ma, C.-H. Leung and W. Wang, A cell-impermeable luminogenic probe for near-infrared imaging of prostate-specific membrane antigen in prostate cancer microenvironments, Eur. J. Med. Chem., 2023, 259, 115659.
107. A. W. Hensbergen, D. M. van Willigen, F. van Beurden, P. J. van Leeuwen, T. Buckle, M. Schottelius, T. Maurer, H.-J. Wester and F. W. B. van Leeuwen, Image-guided surgery: Are we getting the most out of small-molecule prostate-specific-membrane-antigen-targeted tracers?, Bioconjugate Chem., 2019, 31, 375–395.
108. M. Eder, M. Schäfer, U. Bauder-Wüst, W.-E. Hull, C. Wängler, W. Mier, U. Haberkorn and M. Eisenhut, ⁶⁸Ga-complex lipophilicity and the targeting property of a urea-based PSMA inhibitor for PET imaging, Bioconjugate Chem., 2012, 23, 688–697.
109. D. M. Cheff and M. D. Hall, A drug of such damned nature.1 challenges and opportunities in translational platinum drug research, J. Med. Chem., 2017, 60, 4517–4532.
110. W. H. Ang, M. Myint and S. J. Lippard, Transcription inhibition by platinum–DNA cross-links in live mammalian cells, J. Am. Chem. Soc., 2010, 132, 7429–7435.
111. C. S. Burke, A. Byrne and T. E. Keyes, Targeting photoinduced DNA destruction by Ru(II) tetraazaphenanthrene in live cells by signal peptide, J. Am. Chem. Soc., 2018, 140, 6945–6955.
112. L. Blackmore, R. Moriarty, C. Dolan, K. Adamson, R. J. Forster, M. Devocelle and T. E. Keyes, Peptide directed transmembrane transport and nuclear localization of Ru(II) polypyridyl complexes in mammalian cells, Chem. Commun., 2013, 49, 2658–2660.
113. I. Gamba, I. Salvadó, R. F. Brissos, P. Gamez, J. Brea, A. I. Loza, M. E. Vázquez and M. V. López, High-affinity sequence-selective DNA binding by iridium(III) polypyridyl organometallopeptides, Chem. Commun., 2015, 52, 1234–1237.
114. C. Dolan, R. D. Moriarty, E. Lestini, M. Devocelle, R. J. Forster and T. E. Keyes, Cell uptake and cytotoxicity of a novel cyclometalated iridium(III) complex and its octaarginine peptide conjugate, J. Inorg. Biochem., 2013, 119, 65–74.
115. S. Ji, X. Yang, X. Chen, A. Li, D. Yan, H. Xu and H. Fei, Structure-tuned membrane active Ir-complexed oligoarginine overcomes cancer cell drug resistance and triggers immune responses in mice, Chem. Sci., 2020, 11, 9126–9133.
116. M. Fu, X. Han, B. Chen, L. Guo, L. Zhong, P. Hu, Y. Pan, M. Qiu, P. Cao and J. Chen, Cancer treatment: from traditional Chinese herbal medicine to the liposome delivery system, Acta Mater. Med., 2022, 1, 486–506.
117. Y. Hisamatsu, A. Shibuya, N. Suzuki, T. Suzuki, R. Abe and S. Aoki, Design and synthesis of amphiphilic and luminescent tris-cyclometalated iridium(III) complexes containing cationic peptides as inducers and detectors of cell death via a calcium-dependent pathway, Bioconjugate Chem., 2015, 26, 857–879.
118. I. Salvadó, I. Gamba, J. Montenegro, J. Martínez-Costas, J. M. Brea, M. I. Loza, M. V. López and M. E. Vázquez, Membrane-disrupting iridium(III) oligocationic organometallopeptides, Chem. Commun., 2016, 52, 11008–11011.
119. Y. Hisamatsu, N. Suzuki, A.-A. Masum, A. Shibuya, R. Abe, A. Sato, S.-I. Tanuma and S. Aoki, Cationic amphiphilic tris-cyclometalated iridium(III) complexes induce cancer cell death via interaction with Ca²⁺-calmodulin complex, Bioconjugate Chem., 2016, 28, 507–523.
120. K. Yokoi, Y. Hisamatsu, K. Naito and S. Aoki, Design, synthesis, and anticancer activities of cyclometalated tris(2-phenylpyridine)iridium(III) complexes with cationic peptides at the 4′-position of the 2-phenylpyridine ligand, Eur. J. Inorg. Chem., 2017, 2017, 5295–5309.
121. K. Yokoi, C. Balachandran, M. Umezawa, K. Tsuchiya, A. Mitrić and S. Aoki, Amphiphilic cationic triscyclometalated iridium(III) complex–peptide hybrids induce paraptosis-like cell death of cancer cells via an intracellular Ca²⁺-dependent pathway, ACS Omega, 2020, 5, 6983–7001.
122. K. Naito, K. Yokoi, C. Balachandran, Y. Hisamatsu and S. Aoki, Design, synthesis, and anticancer activity of iridium(III) complex-peptide hybrids that contain hydrophobic acyl groups at the N-terminus of the peptide units, J. Inorg. Biochem., 2019, 199, 110785.
123. R. C. Taylor, S. P. Cullen and S. J. Martin, Apoptosis: controlled demolition at the cellular level, Nat. Rev. Mol. Cell Biol., 2008, 9, 231–241.
124. K. Hadian and B. R. Stockwell, The therapeutic potential of targeting regulated non-apoptotic cell death, Nat. Rev. Drug Discovery, 2023, 22, 723–742.
125. A.-A. Masum, K. Yokoi, Y. Hisamatsu, K. Naito, B. Shashni and S. Aoki, Design and synthesis of a luminescent iridium complex-peptide hybrid (IPH) that detects cancer cells and induces their apoptosis, Bioorg. Med. Chem., 2018, 26, 4804–4816.
126. A.-A. Masum, Y. Hisamatsu, K. Yokoi and S. Aoki, Luminescent iridium complex-peptide hybrids (IPHs) for therapeutics of cancer: design and synthesis of IPHs for detection of cancer cells and induction of their necrosis-type cell death, Bioinorg. Chem. Appl., 2018, 2018, 7578965.
127. J. Haribabu, Y. Tamura, K. Yokoi, C. Balachandran, M. Umezawa, K. Tsuchiya, Y. Yamada, R. Karvembu and S. Aoki, Synthesis and anticancer properties of bis- and mono(cationic peptide) hybrids of cyclometalated iridium(III) complexes: Effect of the number of peptide units on anticancer activity, Eur. J. Inorg. Chem., 2021, 2021, 1796–1814.
128. C. Balachandran, K. Yokoi, K. Naito, J. Haribabu, Y. Tamura, M. Umezawa, K. Tsuchiya, T. Yoshihara, S. Tobita and S. Aoki, Cyclometalated iridium(III) complex–cationic peptide hybrids trigger paraptosis in cancer cells via an intracellular Ca²⁺ overload from the endoplasmic reticulum and a decrease in mitochondrial membrane potential, Molecules, 2021, 26, 7028.
129. K. Yokoi, K. Yamaguchi, M. Umezawa, K. Tsuchiya and S. Aoki, Induction of paraptosis by cyclometalated iridium complex-peptide hybrids and CGP37157 via a mitochondrial Ca²⁺ overload triggered by membrane fusion between mitochondria and the endoplasmic reticulum, Biochemistry, 2022, 61, 639–655.
130. W.-Y. Zhang, S. Banerjee, C. Imberti, G. J. Clarkson, Q. Wang, Q. Zhong, L. S. Young, I. Romero-Canelón, M. Zeng, A. Habtemariam and P. J. Sadler, ‘Strategies for conjugating iridium(III) anticancer complexes to targeting peptides via copper-free click chemistry, Inorg. Chim. Acta, 2019, 503, 119396.
131. K. A. Phillips, T. M. Stonelake, K. Chen, Y. Hou, J. Zhao, S. J. Coles, P. N. Horton, S. J. Keane, E. C. Stokes, I. A. Fallis, A. J. Hallett, S. P. O'Kell, J. M. Beames and S. J. A. Pope, Ligand-tuneable, red-emitting iridium(III) complexes for efficient triplet–triplet annihilation upconversion performance, Chem. – Eur. J., 2018, 24, 8577–8588.
132. J. Zhao, K. Yan, G. Xu, X. Liu, Q. Zhao, C. Xu and S. Gou, An iridium(III) complex bearing a donor–acceptor–donor type ligand for NIR-triggered dual phototherapy, Adv. Funct. Mater., 2021, 31, 2008325.
133. Y. Wang, Z. Yi, J. Guo, S. Liao, Z. Li, S. Xu, B. Yin, Y. Liu, Y. Feng, Q. Rong, X. Liu, G. Song, X.-B. Zhang and W. Tan, In vivo ultrasound-induced luminescence molecular imaging, Nat. Photonics, 2024, 18, 334–343.
134. H. J. Knox and J. Chan, Acoustogenic probes: A new frontier in photoacoustic imaging, Acc. Chem. Res., 2018, 51, 2897–2905.
135. G. Li, H. Liu, R. Feng, T.-S. Kang, W. Wang, C.-N. Ko, C.-Y. Wong, M. Ye, D.-L. Ma, J.-B. Wan and C.-H. Leung, A bioactive ligand-conjugated iridium(III) metal-based complex as a Keap1–Nrf2 protein-protein interaction inhibitor against acetaminophen-induced acute liver injury, Redox Biol., 2021, 48, 102129.
136. G. Li, C.-N. Ko, D. Li, C. Yang, W. Wang, G.-J. Yang, C. Di Primo, V. K. W. Wong, Y. Xiang, L. Lin, D.-L. Ma and C.-H. Leung, A small molecule HIF-1α stabilizer that accelerates diabetic wound healing, Nat. Commun., 2021, 12, 3363.

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Footnote

† These authors contributed equally to this work.

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